JP2961182B2 - Pharmaceutical composition for prevention and treatment of Clostridium difficile diarrhea and pseudomembranous colitis - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing and treating Clostridium difficile diarrhea and pseudomembranous colitis.

(Problems to be Solved by Conventional Techniques and Inventions) As humans are born, certain microorganisms in the intestinal tract begin to settle in the intestine and reach 100 trillion to form an almost stable intestinal flora. Speaking of the individual microorganisms that make up the intestinal flora, there may be harm or benefit to the host due to substance production, resolution, etc.
Its involvement in infection prevention and intestinal function (peristalsis, absorption) has been pointed out, and the intestinal flora is deeply involved in host health.

The intestinal flora varies depending on physiological conditions of the host, plants, drugs, stress, and the like. In particular, in recent years, antibacterial agents having excellent antibacterial activity and a broad antibacterial spectrum have been provided in a large amount in a variety of clinical fields, and are used for the treatment and prevention of various infectious diseases. However, the administration of antibacterial agents is the biggest factor affecting the intestinal flora, and by destroying the indigenous flora of the host, it causes abnormal growth of pathogens whose growth is normally suppressed, resulting in diarrhea ,
It is known to cause enteritis and the like. Particularly in human clinical settings, Clostridium difficile (A.
Clostridium difficile diarrhea and pseudomembranous colitis, caused by toxins produced by icilc), are sometimes regarded as serious illnesses that can cause patient death.

In the treatment of Clostridium difficile diarrhea and pseudomembranous colitis, treatment such as discontinuation of administration of the used antimicrobial agent, change, and administration of an intestinal bactericide having a strong antibacterial activity against Clostridium difficile, which is the cause, are treated. Treatment of preceding infections, further disruption of the host intestinal flora due to the antimicrobial properties of the intestinal fungicide itself, new infections under the disturbed flora, dangers of drug resistance of pathogenic bacteria, etc., Clostridium difficile Because spores that are inherently resistant to antibacterial agents are formed in the intestine, they are not easily eliminated by long-term chemotherapy, and after the treatment is stopped, the remaining spores of Clostridium difficile germinate and grow again. If the disease recurs and the patient is significantly debilitated,
In practice, advanced clinical judgment based on a wealth of experience is required, and effective preventive and therapeutic measures are eagerly sought.

Accordingly, an object of the present invention is to provide a novel pharmaceutical composition for preventing and treating Clostridium difficile diarrhea and pseudomembranous colitis.

(Means for Solving the Problems) The above-mentioned object is achieved by a pharmaceutical composition comprising Clostridium difficile diarrhea and pseudocolitis, which is composed of cells or spores of butyric acid bacteria and saccharides.

(Action) By the way, as a new treatment method of this disease, an example has been reported in which butyric acid bacterium was used in a pathological model of diarrhea of Clostridium difficile in golden hamster, a small experimental animal, to prove its effect. The present inventors have found that butyric acid bacteria have antiseptic properties, have close biochemical properties to Clostridium difficile, and are expected to compete for growth in the intestinal tract, and also, butyric acid bacteria do not affect the host flora. This suggests the development of a drug for the prevention and treatment of Clostridium difficile diarrhea and pseudomembranous colitis in humans.
1kg / day adult), acute oral toxicity of butyric acid bacteria in rats, LD50 of _{more than} 5,000mg / kg, preference for administration of dried bacterial powder, differences in intestinal flora in animal species It was not possible due to questions about the validity caused.

Therefore, the present inventors searched the intestinal flora of hamsters and humans, and found that the bacterial density of humans was lower than that of hamsters.
To expect the action of butyric acid bacteria about 10 times higher in humans, I felt the need to increase the concentration of butyric acid bacteria in the intestine. As a countermeasure, Clostridium butyricum MIYAIRI 5 is a kind of butyric acid bacterium.
88) (abbreviated as CbM) The bacterial paste was kneaded with cornstarch, a saccharide that is easy for the strain to use as a carbon source and is not directly absorbed from the intestinal tract, to prepare a dried powder. The kneading ratio of CbM and corn starch is fixed, but the antimicrobial agent is administered as "Miya BM" (kneading 40 mg of CbM powder into 960 mg of corn starch, 1 g of CbM spores and containing 10 ⁸ CFU in CgM). When administered to humans and examined for recovery from feces, even a small dose (10 ⁶ CFU / kg) showed a higher recovery (10 ⁷ CFU / g feces) compared to the hamster experiment. It was inferred that the high concentration of CbM was achieved. The powder had almost no odor peculiar to CbM, and kneading of corn starch also contributed to palatability. Next, fecal search was performed before administration of the antimicrobial agent in hospitalized patients who received antimicrobial therapy, without any notable diseases of the gastrointestinal tract.
M powder was divided into a group that received 1 g of the powder 1 to 3 times a day and a group that did not receive the powder. Subsequently, the feces were searched. In the CbM-administered group, Clostridium difficile or its toxin was detected in the feces. Patients account for 5% of CbM recipients,
Compared with 30% of the non-administration group, CbM administration was significantly effective in suppressing the growth of Clostridium difficile in human intestine. In addition, complaining of abdominal symptoms after administration of an antibacterial agent, and giving "Miya BM" as a CbM powder in a dose of 1 g 1 to 3 times a day to an inpatient in which Clostridium difficile is detected in the feces, Symptoms and stool properties were improved, and Clostridium difficile in feces was culture-negative on day 10 of administration of "MIYA BM", leading to the completion of the invention. CbM is an anaerobic spore bacterium, under anaerobic conditions,
Proliferates and spores in common media. For example, ordinary broth with 2% corn starch is suitable.

As the saccharide used in the present invention, lactic acid bacteria can be used, and any of monosaccharides, disaccharides and polysaccharides can be used, but polysaccharides are preferable. Monosaccharides include xylose, arabinose, glucose, mannose, fructose and the like. Disaccharides include sucrose, maltose, cellobiose, lactose and the like. Examples of the polysaccharide include starch, dextrin, glycogen, pectin, raffinose, amygdalin, glycomannan, etc., and particularly raffinose, starch and the like. These sugars are
1 mg to 10 g is mixed with 10 ⁸ CFU of butyric acid bacteria.

The pharmaceutical compositions according to the invention are administered 10 ⁶ to 10 ⁹ CFU as cell or spores of human body weight 1kg per butyric acid bacteria. The composition is orally administered with a pharmaceutically acceptable carrier, if necessary.

A butyric acid bacterium refers to a microorganism that produces butyric acid as a metabolite thereof, and is particularly preferably Clostridium butyricum.

As antibacterial agents, penicillins (Penicillins) cephalosporins (Cephalosporins), aminoglycosides (Aminoglycosides), macrolides (Macrolides),
Lincosamides (Lincosamides), tetracycline (Te
tracyclines), peptides (Peptides), depsipeptides, Rifamycin group (Rifamycin)
s), Sulfonamides, Pyridon carboxylic acids, Nitrofurans, Nitroimdazole compounds, Antituberculous a
gents), antitumor substances (Antineoplastic agents), antifungal substances (Antimycotics), trimethoprim (Trimethoprim)
prim), chloramphenicol,
Novobiocin, fusidic acid (Fusidic ac)
id), Fosfomycin, Cycloserine The pharmaceutical compositions according to the invention have very low acute and chronic toxicity.

(Examples) Hereinafter, the present invention will be described in detail with reference to examples. Clostridium, butyricum, and Miyairi used in the experiment
588 (Clostridium butyricum MIYAIRI 588) is stored in the National Institute of Microbial Technology under the accession number No. 2789.

Example 1 Feces of 15 golden hamsters (70 g, male) and feces of 69 patients who had no gastrointestinal illness and had not received antimicrobial therapy were administered intestines according to a generally known method. The internal flora was searched. As shown in Table 1, anaerobic bacteria were particularly dominant in the human intestinal flora, and it was found that the bacterial density per gram of feces reached 10 times that of hamster.

Example 2 Five golden hamsters (70 g, male) were each treated with cefatridine (Cefatr), one of cephem antibiotics.
izine) (abbreviated as CFT) at a concentration of 5.25 mg was suspended in purified water and orally administered only once using a sonde. Then, CbM-dried bacteria containing 10 ⁹ CFU / day of CbM spores per animal were suspended. 1 ml of powder
Rectal stool on the 5th day of administration, which was suspended in purified water 3 times a day and given orally, and "Miya BM" (CbM bacterial powder 40 mg)
Kneaded into 960 mg of corn starch, and spores of CbM
Includes ⁸ CFU. ) Was administered as a CbM powder at a dose of 1 g once to three times a day. The patient received antimicrobial therapy (penicillin group, cephalosporin group antibiotics) without any noticeable disease in the gastrointestinal tract. The number of viable CbM bacteria in feces was measured for feces of five patients. CbM isolation culture is 5% horse blood BL for CbM
Agar medium (Cycloserine 350mg, Kanamycin 1mg, Clindamycin (Clindamy)
(cin) 75 μg and Trimethoprim 12 mg were added to the medium 1 respectively. ) Using anaerobic culture (37
° C, 72 hours). As a result, as shown in Table 2, CbM was detected from all the 10 samples. "Miya BM" for humans CbM
The recovered CbM concentration when administered as a powder was higher than when CbM bacterial powder was administered to hamsters, and the administration efficiency in terms of the dose per body weight was higher when `` Miya BM '' was administered to humans as hamster powder. It is thought that the growth of CbM was promoted for some reason, such as that corn starch was a good carbon source for CbM by bringing corn starch into close contact with the CbM spores and sending it into the intestine. The fecal flora reflects the intestinal flora, and it is considered that a CbM preparation obtained by kneading CbM and corn starch is required as an oral drug to expect the action of CbM in the human intestinal tract.

Example 3 For inpatients receiving antimicrobial therapy without any noticeable diseases of the gastrointestinal tract, they received feces and antimicrobial therapy (penicillin group, cephalosporin group antibiotics) before administration of antimicrobial agents. After that, those inpatients were referred to as “Miya BM” (CbM powder 40 mg).
Kneaded into 960 mg corn starch, and CbM spores are added to 1 g at 10 ⁸ CFU
Including. ) Was administered as a CbM powder, and a group (20 subjects) and a group (20 subjects) who received 1 g of a dose 1 to 3 times a day were tested for the detection of Clostridium difficile and its toxin in feces, respectively. The method was to detect Clostridium difficile by anaerobic culture (37 ° C, 72 ° C) using CCFA medium.
The detection limit for hr and culture was 400 CFU / g (feces). )
The detection of Clostridium difficile toxin
Latex reagent [Clostridium difficile D-
Detection of one toxin is possible at 500ng / ml (sample). ]. As shown in Table 3, in the CbM-administered group, 5% of CbM-administered patients detected Clostridium difficile or its toxin in feces, and CbM-administered in humans compared with 30% in the non-administered group. In the growth inhibition of Clostridium difficile. The properties of the clinical isolates of Clostridium difficile were as shown in Table 4.

Example 4 Among inpatients who received antimicrobial therapy (ampicillin for oral use) without any noticeable disease in the gastrointestinal tract, two inpatients whose Clostridium difficile was detected in their faeces were " MIYA BM ”as CbM powder 1g 1
Give 1 to 3 times a day. As shown in Table 5, the stool properties were improved in both subjects as shown in Table 5. Clostridium difficile in feces showed culture negative on day 10 of administration of "Miya BM", and Cb
M was detected in feces. In addition, the abdominal symptoms that were initially complained disappeared within two to three days after administration of "Miya BM". The characteristics of clinical isolates of C. difficile are:
Table 6 shows the results.

(Effect of the Invention) As described above, the present invention is a pharmaceutical composition for preventing and treating Clostridium difficile diarrhea and pseudomembranous colitis comprising cells or spores of butyric acid bacteria and saccharides. The administration has the advantage that the above symptoms are prevented or treated and the toxicity is extremely low.

Claims (1)

(57) [Claims] 1. A pharmaceutical composition comprising Clostridium difficile diarrhea and pseudomembranous colitis, comprising a cell or spore of butyric acid bacteria and a saccharide.